Cysteamine with In Vitro Antiviral Activity and Immunomodulatory Effects Has the Potential to Be a Repurposing Drug Candidate for COVID-19 Therapy

The ongoing pandemic of coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), needs better treatment options both at antiviral and anti-inflammatory levels. It has been demonstrated that the aminothiol cysteamine, an already human applied drug, and its disulfide product of oxidation, cystamine, have anti-infective properties targeting viruses, bacteria, and parasites. To determine whether these compounds exert antiviral effects against SARS-CoV-2, we used different in vitro viral infected cell-based assays. Moreover, since cysteamine has also immune-modulatory activity, we investigated its ability to modulate SARS-CoV-2-specific immune response in vitro in blood samples from COVID-19 patients. We found that cysteamine and cystamine decreased SARS-CoV-2-induced cytopathic effects (CPE) in Vero E6 cells. Interestingly, the antiviral action was independent of the treatment time respect to SARS-CoV-2 infection. Moreover, cysteamine and cystamine significantly decreased viral production in Vero E6 and Calu-3 cells. Finally, cysteamine and cystamine have an anti-inflammatory effect, as they significantly decrease the SARS-CoV-2 specific IFN-γ production in vitro in blood samples from COVID-19 patients. Overall, our findings suggest that cysteamine and cystamine exert direct antiviral actions against SARS-CoV-2 and have in vitro immunomodulatory effects, thus providing a rational to test these compounds as a novel therapy for COVID-19.


Introduction
Coronavirus disease 2019 (COVID- 19), which is caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global concern due to its high transmissibility and pathogenicity [1]. While great efforts have been focused on vaccine development, many unanswered questions remain regarding the development of <18 years of age. All patients provided informed written consent. Demographic and clinical information were collected at enrollment.

Virus Protection Assay
The antiviral activity of cysteamine, cystamine, or Z-DON was tested by cytopathic effects (CPE) inhibition assay using Vero E6 cells. The determination of the in vitro susceptibility of SARS-CoV-2 to those compounds is expressed as percentage of surviving cells, as we recently described [27]. Briefly, Vero E6 cells were washed with 1× PBS, dislodged with trypsin-EDTA solution (Merck Life Science, Cat. No. T3924), and seeded into 96-well imaging plates at a density of 2.5 × 10 4 cells per well in culture medium, as indicated above. The cells were incubated for 24 h to allow adherence. Cells were treated 1 h before SARS-CoV-2 infection with either a 1:2 serial dilution of cysteamine, cystamine, or H 2 O as control or with 1:3 serial dilution of Z-DON or DMSO as control. Cells were infected at 0.001 multiplicity of infection (MOI; which reflects the ratio of PFU to the number of infected cells) using MEM supplemented with heat-inactivated 2% FBS and 2 mM L-glutamine in the presence of the different treatments. After 1 h incubation, the viral input was replaced by fresh medium containing the different compounds/controls. Cells were then treated every 24 h by adding the drugs to the culture medium, incubated at 37 • C with 5% CO 2 for 72 h, and cell viability was evaluated by a standard crystal violet staining assay and measuring the optical density (OD) at 595 nm. Results were analyzed using GraphPad Prism 7.04 (GraphPad, San Diego, CA, USA) and reported as the percentage of surviving cells compared to the uninfected cells.
In some experiments, cells were treated using different protocols in terms of time of drug administration in respect to SARS-CoV-2 infection. In particular, Vero E6 cells were: (i) pre-treated for 1 h and then infected for 1 h in the absence of drugs (MOI = 0.001) for pre-treatment protocol; (ii) infected for 1 h in the presence of drugs (MOI = 0.001) for co-treatment protocol; and (iii) infected for 1 h in the absence of drugs (MOI = 0.001), virus removed and drugs added at 1 h.p.i. for post-treatment protocol. Afterwards, the virus was removed and the cells were cultured in the presence of compounds that were added 1 h, 24 h, and 48 h after SARS-CoV-2 infection, as described above, until the end of the experiments (72 h.p.i.).

SARS-CoV-2 Virus Yield Assay
The ability of the test compounds to reduce the SARS-CoV-2 replication was explored by a typical virus yield reduction assay. The virus strain isolated at INMI L. Spallanzani IRCCS (2019-nCoV/Italy-INMI1; GenBank MT066156) [28] was used in all the experiments performed with the different cell lines (Vero E6, Calu-3, or Huh7). Virus replication in the various experimental conditions was measured by back-titrating the culture supernatants of the infected cells by limiting dilution assay in Vero E6 cells, as we recently described [27]. Readout of the virus back-titration was based on detection of CPE, and infectious titer was expressed as 50% tissue-culture effective dose (TCID50) values, calculated according to the Reed-Muench method.

Viral RNA Quantification
The viral RNA present in the culture medium of the samples described above were quantified by Ct measurement with Simplexa™ COVID-19 Direct assay (DiaSorin, Vicenza, Italy), as previously described [29] according to the manufacturer's instructions. Briefly, 50 µL of culture medium and 50 µL of Reaction Mix were added to their specific wells on a direct amplification disk, which was loaded onto the LIAISON ® MDX instrument (Dia-Sorin).

Transmission Electron Microscopy
Transmission electron microscopy (TEM) was performed on cultured cells using standard procedures. Cultured cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, for 4 h at 4 • C. Post-fixation was performed with 1% OsO4. Samples were then dehydrated in graded ethanol and embedded in Epon resin, as previously described [30,31]. Ultrathin sections were stained with 2% uranyl acetate and observed under a transmission electron microscope (JEOL JEM 2100 Plus, Japan Electron Optics Laboratory Co. Ltd., Tokyo, Japan). Images were captured digitally with a digital camera TVIPS (Tietz Video and Image Processing Systems GmbH, Gauting, Germany). Quantitative analysis of infected cells was assessed in blind by two authors counting 100 cells per condition.

Immunoblotting Analysis
Western blotting analysis was carried out as previously reported [32]. Whole cell extracts (

Statistical Analysis
Data were analyzed using GraphPad Prism 7.04 (GraphPad) using Wilcoxon matchedpairs rank test. Medians and interquartile ranges (IQR) were calculated for continuous measures; the Friedman test was used for comparisons among groups and the Wilcoxon matched-pairs rank test with Bonferroni correction for pairwise comparisons.

Cysteamine and Cystamine Significantly Reduce the Cytopathic Effect Induced by SARS-CoV-2 in Vero E6 Cells
We characterized the impact of cysteamine and cystamine on SARS-CoV-2 by evaluating their effect on the virus-induced cytopathic effect (CPE) in Vero E6 cells. Cells were treated with either cysteamine or cystamine using two-fold serial dilutions of compounds ranging from 2000 µM to 31.25 µM, or with the vehicle [H 2 O from 2% to 0.031% (v/v)] as the control.
Firstly, we assessed the safety of cysteamine or cystamine treatment of Vero E6 cells, and found that high concentrations of both compounds had toxic effects, with a 50% cytotoxic concentration (CC 50 ) of 660 ± 243 µM ( Figure 1A) and 357 ± 68 µM, respectively ( Figure 1B). Therefore, for the CPE assay we considered only the non-toxic concentrations with ranges of 500-31.25 µM for cysteamine and 250-31.25 µM for cystamine. As shown in Figure 1C and D, both compounds significantly prevented the SARS-CoV-2-induced CPE in a dose-dependent manner. In particular, cysteamine significantly reduced the CPE at 500 µM and 250 µM compared to drug-vehicle (H 2 O) ( Figure 1C), with a 50% inhibitory concentration (IC 50 ) value of 180 ± 54 µM. On the other hand, cystamine significantly reduced the CPE from 250 µM to 31.25 µM with an IC 50 value of 81 ± 39 µM ( Figure 1D).

Cysteamine and Cystamine Significantly Reduce SARS-CoV-2 Production in Vero E6 Cells
We evaluated whether these compounds directly impact the SARS-Cov-2 life cycle, measuring the amount of the infectious viral particles released in the culture medium at 72 h post-infection of cells treated with cysteamine (500 µM, 250 µM and 125 µM), cystamine (250 µM, 125 µM and 62.5 µM) or with the drug-vehicle (H 2 O). Notably, cysteamine significantly reduced the yield of infectious SARS-CoV-2 mainly when used at 500 µM ( Figure 1E), while cystamine significantly reduced viral yield at all the concentrations tested ( Figure 1F).   We also quantified viral RNA present in the culture medium by qRT-PCR. As shown in Supplementary Figure S1, a significant reduction of viral RNA was found only at the higher concentration tested of either cysteamine or cystamine (panels A and B, respectively), thus indicating that non-infectious RNA is present in culture supernatants. We also measured viral RNA by time-course experiments treating cells with the maximum tolerated concentration of cysteamine or cystamine (500 µM or 250 µM, respectively). The inhibitory effect of viral production mediated by the drugs was already evident at 24 h.p.i. and increased by the time of culture (Supplementary Figure S1C,D).
We also performed a transmission electron microscopy analysis of Vero E6 cells at 48 h.p.i., before the virus-mediated CPE was evident. Without treatment, 100% of cells showed a great number of viral particles, whereas drug treatment reduced it up to only 34% (Supplementary Figure S2B). In the untreated infected cells, we observed the presence of large amounts of viral particles along the cell surface (Figure 2A,B). Virus particles showed the characteristic black dots, due to cross-section through the viral nucleocapsid, while spikes were not always visible ( Figure 2B inset). Infected cells became round in shape and displayed several intracellular changes. Ribosomes appear often grouped rather than randomly diffuse in the cytoplasm, and a higher number of vacuoles are observed, many of them consisting of lipolysomes ( Figure 2A). SARS-CoV-2 particles were enclosed in membrane-bound vacuoles with different size and shape. Some of these vacuoles also contained membrane wraps and electron dense lipid materials ( Figure 2B). On the other hand, the majority of infected cells treated with cysteamine (500 µM) displayed a regular morphology, with normal elongated shape and well preserved cytoplasmic organelles with no signs of virus presence ( Figure 2C,D). In cysteamine-treated cells we found rare viral particles and a lower number of intracytoplasmic vacuoles containing virions (Supplementary Figure S2A). Importantly, in those cells we found the characteristic change associated to virus presence such as the lipolysosomes. Cells not infected and treated with cysteamine alone did not show signs of ultrastructural damage and were used as internal controls (Supplementary Figure S3).
Overall, cysteamine and cystamine significantly decreased SARS-CoV-2 replication, as shown by a decrease of the viral-induced CPE and by the analysis of the electron microscopy images.

Cysteamine and Cystamine Significantly Reduce SARS-CoV-2-Induced CPE in Vero E6 Cells, Independent of the Time of Treatment
It has been shown that cysteamine and several other thiol-based compounds decrease the binding of the spike protein to ACE2 receptor [21]. Therefore, here, we evaluated whether the inhibition of virus replication by cysteamine or cystamine was exerted by inhibiting the viral entry into Vero E6 cells. To this aim, different protocols of drug treatments were used: (i) 1 h before infection (pre-treatment); (ii) concomitantly with the infection (co-treatment); and (iii) 1 h after infection (post-treatment).
As shown in Figure 3, no significant differences in the extent of the CPE inhibition were found between the pre-treatment regimen, used as a reference (100% of CPE inhibition activity), and either the co-treatment or post-treatment protocols when cysteamine ( Figure 3A) or cystamine ( Figure 3B) were used. Overall, our results suggest that these compounds exert their antiviral activity through cellular-mediated mechanisms rather than inhibiting viral entry.  TG2-KO cells and their control (Huh7-CTR), were infected with SARS-CoV-2 (MOI = 1 and MOI = 0.1) for 48 h, when the culture medium was collected and used for back-titration of virus yield. No significant differences were found in the viral production of Huh7-TG2-KO and Huh7-CTR cells (Supplementary Figure S4C), thus demonstrating that TG2 does not have any effect on SARS-CoV-2 replication.

Cysteamine and Cystamine Significantly Reduce SARS-CoV-2 Production in Calu-3 Cells
We next tested whether cysteamine and cystamine exert their antiviral activity in the lung-derived epithelial Calu-3 cells, as these cells are relevant as a model for human infections. Since SARS-CoV-2 did not induce CPE on Calu-3 cells, we evaluated viral production measuring the infectious virus yield (TCID50). Cells were pre-treated for 1h with Since cysteamine is commonly used as a TG2 inhibitor [10,13,14], we investigated whether TG2 could play a role on SARS-CoV-2 replication. Firstly, we used Z-DON, a specific inhibitor of the transamidating activity of TG2 [13], in the CPE inhibition assay in Vero E6 cells. Interestingly, Z-DON did not show any inhibition of the SARS-CoV-2-induced CPE (Supplementary Figure S4B). We also analyzed the viral production of Huh7 cells, a hepatoma cell line known to support SARS-CoV-2 replication [33], in which the TG2 genes have been deleted with CRISPR/Cas9 technology (Huh7-TG2-KO). Since SARS-CoV-2 did not induce CPE on this cell line, we evaluated the viral production measuring the infectious virus yield (TCID 50 ), as described above using Vero E6 cells. Huh7-TG2-KO cells and their control (Huh7-CTR), were infected with SARS-CoV-2 (MOI = 1 and MOI = 0.1) for 48 h, when the culture medium was collected and used for back-titration of virus yield. No significant differences were found in the viral production of Huh7-TG2-KO and Huh7-CTR cells (Supplementary Figure S4C), thus demonstrating that TG2 does not have any effect on SARS-CoV-2 replication.

Cysteamine and Cystamine Significantly Reduce SARS-CoV-2 Production in Calu-3 Cells
We next tested whether cysteamine and cystamine exert their antiviral activity in the lung-derived epithelial Calu-3 cells, as these cells are relevant as a model for human infections. Since SARS-CoV-2 did not induce CPE on Calu-3 cells, we evaluated viral production measuring the infectious virus yield (TCID50). Cells were pre-treated for 1 h with different doses of cysteamine, cystamine or H 2 O as control before SARS-CoV-2 infection (MOI = 0.1). Cells were then cultured in the presence of the drugs that were added 1 h and 24 h after infection until the end of the experiments (48 h.p.i.), when the culture medium was collected and viral titers measured. As shown in Figure 4, cysteamine and cystamine significantly reduced viral production in Calu-3 cell cultures, thus suggesting that these compounds exert a potent direct antiviral effect also in human cells, which are similar to the natural target of SARS-CoV-2.
24 h after infection until the end of the experiments (48 h.p.i.), when the culture medium was collected and viral titers measured. As shown in Figure 4, cysteamine and cystamine significantly reduced viral production in Calu-3 cell cultures, thus suggesting that these compounds exert a potent direct antiviral effect also in human cells, which are similar to the natural target of SARS-CoV-2. Absorption of the virus was allowed for 1 h at 37 °C in the presence of different treatments. The unabsorbed virus was removed and replaced by fresh medium with cysteamine, cystamine, or H2O. Cells were then treated every 24 h and incubated at 37 °C with 5% CO2 for 48 h when culture medium was collected. Virus yield was measured by back-titration of supernatants as described in the methods. Virus titers are expressed as 50% tissue-culture effective dose (TCID50/mL) values according to the Reed-Muench method. Statistically significant differences between compound-and H2Otreated cells have been calculated using Wilcoxon matched-pairs rank tests.

Cysteamine and Cystamine Decrease the SARS-CoV-2-Specific Response in COVID-19 Patients
It is known that a dysregulated immune response is associated with COVID-19 disease severity [34], which is the rational for the use of drugs such as dexamethasone [35,36] or baricitinib [37][38][39] for COVID-19 treatment. Cysteamine has been shown to have antiinflammatory properties [15][16][17]. Therefore, we evaluated whether cysteamine or cystamine had an immunomodulatory effect on the SARS-CoV-2-specific immune response. In particular, we evaluated the impact of these drugs on the in vitro IFN-γ-viral-specific response in the whole blood platform, as described [22,23,26]. Demographic and clinical characteristics of the 26 enrolled subjects are shown in Table 1. To better address the effect of these two compounds on the response to SARS-CoV-2 antigens (spike, membrane, and nucleocapsid), we analyzed the IFN-γ response considering only the IFN-γ values >0 pg/mL [22]. The analysis was, therefore, conducted on 24 patients for pool S ( Figure 5A Cysteamine or cystamine significantly decreased the IFN-γ levels in response to pool S (pool S median: 44.6 pg/mL, IQR: 12.0-128.6 pg/mL; pool S+cysteamine median: 33.6 pg/mL, IQR: 10.8-86.9; pool S+cystamine median: 37.7, IQR: 10.4-94.9) compared to the untreated control (p = 0.009 and p = 0.007, respectively) ( Figure 5A). Figures 5B and 5C show the impact of cysteamine or cystamine on the IFN-γ production in stimulated whole Cells were then treated every 24 h and incubated at 37 • C with 5% CO 2 for 48 h when culture medium was collected. Virus yield was measured by back-titration of supernatants as described in the methods. Virus titers are expressed as 50% tissue-culture effective dose (TCID 50 /mL) values according to the Reed-Muench method. Statistically significant differences between compound-and H 2 O-treated cells have been calculated using Wilcoxon matched-pairs rank tests.

Discussion
In this study, we demonstrated that cysteamine and its disulfide product of oxidation cystamine display potent in vitro antiviral activity against SARS-CoV-2 and ex vivo immunomodulatory properties in blood cells from COVID-19 patients. These findings are particularly relevant since cysteamine is an approved drug routinely used as a gold standard for the treatment of cystinosis, a rare inherited autosomal recessive disease. Indeed, using different cell lines, we showed that treatment with cysteamine or cystamine exerted antiviral activity by impairing SARS-CoV-2 replication, as clearly highlighted by the presence of very few viral particles in the infected cells upon cysteamine treatment in the electron microscopy images. Moreover, drug treatment almost eliminated the typical cytopathic effects observed in untreated SARS-CoV-2-infected cells (i.e. rounded shape, grouped ribosomes, vacuolar degeneration consisting of accumulation of lipolysomes).
Interestingly, since the post-infection treatment has the same antiviral properties as the pre-infection treatment, the effects elicited by cysteamine or cystamine are likely due to the intracellular antiviral activity of the drugs and not to the reduced binding of the spike protein to the ACE2 receptor, as suggested for several thiol-based compounds [21]. Considering the antioxidant and immune-modulatory activities of cysteamine or cystamine [15][16][17], we evaluated the impact of these drugs on the in vitro SARS-CoV-2-specific response in blood from COVID-19 patients. The specific response was significantly reduced either to pool M or pool S, as well as upon SEB stimulation, indicating that cysteamine, in addition to the direct antiviral action, has also a potent immunomodulatory activity. This finding is particularly relevant considering that a dysregulated immune response is one of the hallmarks of COVID-19 characterized also by the so-called "cytokine storm" present in patients with severe disease. Indeed, it has been reported that aberrant pathogenic Th1 cells with co-expressing IFN-γ and GM-CSF are present in patients with severe disease compared to those with milder disease, suggesting that pathogenic Th1 cells may play a critical role in hyper-inflammatory responses during Covid-19 pathogenesis [40]. The role of type II IFN is currently poorly understood; it may be proviral in some sites by inducing expression of the viral receptor and may also drive inflammation. Indeed, treatment with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection [41]. Furthermore, the percentage of IFN-γ-producing CD4 + T cells has been found increased in extremely severe patients, as well as the CD8 + T cells, thus indicating that the hyperfunction of these T cells could be associated with the pathogenesis of extremely severe SARS-CoV-2 infection [42] or merely with a specific response [43]. Current routine therapies are crucial for reducing the cytokine storm and are widely used depending on the disease stage of the patients [37,38].
Since treatment with cysteamine and cystamine may influence many intracellular and extracellular processes, it is difficult to draw definitive conclusions on the mechanism(s) responsible for the observed results. In fact, their free thiol group is highly reactive and can react with free thiol or the disulfide bounds of proteins, possibly interfering with their function such as, for instance, the cysteine protease caspase-3 or the tyrosinase dopaoxidase [44,45]. Cysteamine promotes the transport of cysteine into cells promoting the glutathione synthesis, a potent intracellular antioxidant. It is known that the redox cellular state plays a key role in the regulation of many essential signaling pathways, including cell death and proliferation, and modulates the expression of several redox-sensitive genes.
Cysteamine and cystamine have been used as transglutaminase inhibitors. In particular, cysteamine inhibits transglutaminase by acting as a competitive inhibitor for the transamidation reactions catalyzed by this enzyme. Factor XIII (FXIII) is a plasma transglutaminase that crosslinks fibrin monomers and alpha(2)-plasmin inhibitor during clot formation; deficiency of FXIII worsens clot stability and increases bleeding. Interestingly, it has been shown that the transamidating activity of FXIIIa required for the proper clot stabilization is inhibited by cysteamine [46], thus the multifunctional effects of these drugs could also be beneficial in preventing the thrombosis associated with SARS-CoV-2 infection. In fact, a key aspect of the pathophysiology of COVID-19 is related to the hypercoagulable and hypofibrinolytic state observed in patients affected by severe disease, supported by the evidence of elevated D-dimer that represents the sign of excessive activation of coagulation. Many clinical data indicate a general beneficial effect of heparin for hospitalized COVID-19 patients, leading to a reduced mortality in subjects with markedly elevated D-dimer or high fibrinogen levels. Hence, the beneficial effects of cysteamine and cystamine during SARS-CoV-2 infection may exceed those observed in this study.
Although some scientific findings suggest that autophagy plays an important role in viral replication and pathogenesis, its role during SARS-CoV-2 infection remains to be completely defined. In keeping with this notion, we showed that cysteamine impairs the bacterium replication in Mtb-infected macrophages by inhibiting autophagy in vitro [14]. Thus, it would be important to explore in the future whether the cysteamine effects on SARS-CoV-2 are related to the modulation of cell autophagy, and how these events affect the host cell antiviral properties.
In conclusion, our findings indicate that cysteamine and cystamine modulate both viral replication and SARS-CoV-2-mediated immune response in vitro. These results are interesting to be further confirmed and expanded to animal models and humans as a new drug for COVID-19 treatment. Considering that cysteamine is a drug approved by the FDA and EMA, it would be important to conduct clinical trials to verify the therapeutic effect of these small molecules in COVID-19 patients. In particular, specifically designed clinical trials should validate the safety and the efficacy of cysteamine in clinical practice when used alone or in combination with other approved drugs.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of National Institute for Infectious Diseases (INMI) "Lazzaro Spallanzani" (approval number 59/2020).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The raw data generated and/or analyzed within the present study are available in our institutional repository (rawdata.inmi.it), subject to registration. The data can be found by selecting the article of interest from a list of articles ordered by year of publication. No charge for granting access to data is required. In the event of a malfunction of the application, the request can be sent directly by e-mail to the Library (biblioteca@inmi.it).